Research in our group explores the mechanisms of genome stability,
including the biochemical mechanism repairing cross-links that form
between DNA strands and the DNA polymerases that help cells tolerate
DNA damage, as well as their consequences for cancer.
The panel at the right shows chromosomes from mouse skin keratinocytes, either with an intact Rev3l (+/lox) or inactivated Rev3l (-/lox) gene. Rev3l is one of the genes necessary to defend cells against DNA crosslinks. The Rev3l gene encodes the catalytic subunit of DNA polymerase ζ.
This area of our research examines the biochemical mechanism of repair of cross-links between DNA strands. Chemicals that produce cross-links are particularly destructive to genome function and consequently, are widely used in cancer chemotherapy.
Exogenous sources of intra-stand crosslinking chemicals include mitomycin C, nitrogen mustard, cisplatin, and psoralen. Endogenous sources creating crosslinks include metabolites created by lipid peroxidation such as acrolein, crotonaldehyde, malondialdehyde and nitric oxide. These agents are widely used for the treatment of leukemia and solid tumors. Although the mechanisms mammalian cells use to repair intra-strand crosslinks are not well understood, the nucleotide excision repair (NER) pathway may be highly involved in this process. We study how NER proteins help repair DNA crosslinks.
1P01 CA193124 NIH/NCI “Processing and Repair of DNA Crosslinks”
(Wood, PI) 02/01/2017 – 01/31/2022
Wang R, Lenoir WF, Wang C, Su D, McLaughlin M, Hu Q, Shen X, Tian Y, Klages-Mundt N, Lynn E, Wood RD, Chen J, Hart T, Li L. DNA polymerase iota (ι) compensates for Fanconi anemia pathway deficiency by countering DNA replication stress. Proc Natl Acad Sci U S A. 2020 Dec 29;117(52):33436-33445.
Manandhar M, Lowery MG, Boulware KS, Lin KH, Lu Y, Wood RD. Transcriptional consequences of XPA disruption in human cell lines. DNA Repair (Amst). 2017 Sep;57:76-90.
FAM35A and DNA Resection
We identified the previously uncharacterized protein FAM35A as a REV7-associated factor. FAM35A contains OB-fold domains suggesting that it binds to single-stranded DNA. Our experiments indicate that FAM35A participates in controlling resection of DNA ends, and several other research groups have identified FAM35A and REV7 as components of the “Shieldin” complex. We found that FAM35A is absent in a BRCA1-mutant tumor (HCC1937) with anomalous resistance to PARP inhibitors. The FAM35A gene is deleted frequently in prostate cancers.
Reference: Tomida J, Takata Ki, Bhetawal S, Person MD, Chao HP, Tang DG, Wood RD. FAM35A associates with REV7 and modulates DNA damage responses of normal and BRCA1-defective cells EMBO J. 2018;37(12):e99543.
Active site of DNA polymerase theta
DNA Polymerase theta (POLQ)
We have also identified and are studying the function of other DNA processing enzymes including DNA polymerase theta. We cloned the mammalian gene and found that it has a role in a specific DNA double-strand break (DSB) repair mechanism, theta-mediated end-joining (TMEJ). TMEJ relies on an overlap between very short regions of identical DNA sequence, called microhomologies, on single-stranded DNA. As these microhomologies are not usually at the very ends of the single-stranded DNA, repair requires another enzymatic activity to remove the unmatched parts of the single-stranded DNA ends. In most forms of double-strand break repairs, this trimming step requires a separate nuclease, but in an unprecedented discovery, made with our collaborator Dr. Sylive Doublié of the Larner College of Medicine at University of Vermont, we found that DNA polymerase θ acts not only as a polymerase, but also as an endonuclease, and that the same active site controls these dual functions. Further, the end-trimming function of POLQ is inhibited by dideoxynucleotides, suggesting that chain-terminating nucleoside analogs could be clinically useful as POLQ inhibitors.
NIH grants: P01 CA193124 and P01 CA247773
Zahn KE, Jensen RB, Wood RD*, Doublié S*. Human DNA polymerase theta harbors DNA end-trimming activity ctirical for DNA repair. Mol Cell. In press.
Carvajal-Garcia J, Cho JE, Carvajal-Garcia P, Feng W, Wood RD, Sekelsky J, Gupta GP, Roberts SA, Ramsden DA. Mechanistic basis for microhomology identification and genome scarring by polymerase theta. Proc Natl Acad Sci U S A. 2020 Apr 14;117(15):8476-8485. doi: 10.1073/pnas.1921791117. Epub 2020 Mar 31. PMID: 32234782
Feng W, Simpson DA, Carvajal-Garcia J, Price BA, Kumar RJ, Mose LE, Wood RD, Rashid N, Purvis JE, Parker JS, Ramsden DA, Gupta GP. Genetic determinants of cellular addiction to DNA polymerase theta. Nat Commun. 2019 Sep 19;10(1):4286. doi: 10.1038/s41467-019-12234-1. PMID: 31537809
Malaby AW, Martin SK, Wood RD, Doublié S. Expression and Structural Analyses of Human DNA Polymerase θ (POLQ). In: Eichman B, editor. DNA Repair Enzymes: Structure, Biophysics, and Mechanism. Methods in Enzymology. 2017;592:103-121
Yousefzadeh MJ, Wyatt DW, Takata K, Mu Y, Hensley SC, Tomida J, Bylund GO, Doublié S, Johansson E, Ramsden DA, McBride KM, Wood RD. Mechanism of suppression of chromosomal instability by DNA polymerase POLQ. PLoS Genet. 2014 Oct 2;10(10):e1004654.
Rev3l knockout leads to abnormal skin morphology
DNA polymerase zeta (pol ζ) is exceptionally important for controlling mutagenesis and genetic instability in cells and is comprised of two proteins, REV3L and REV7. REV3L is the catalytic subunit of pol ζ, whereas REV7 (MAD2L2) is considered an accessory subunit.
We have generated mouse models with specific deficiencies in DNA polymerase zeta. These models reveal that pol ζ is critical for maintaining chromosome stability and limiting tumorigenesis. Deletion of Rev3L in p53 mutant mice leads to increased cancer incidence and a shortened lifespan. Further, two distinct REV7-binding sites in REV3L are needed to prevent chromosome breaks and confer resistance to DNA damage.
Martin SK, Wood RD. DNA polymerase zeta in DNA replication and repair. Nucleic Acids Res. 2019 Sep 19;47(16):8348-8361.
Lange SS, Bhetawal S, Reh S, Powell KL, Kusewitt D, Wood RD. DNA polymerase ζ deficiency causes impaired wound healing and stress-induced skin pigmentation. Life Science Alliance. 2018 June 29; 1(3):e201800048.
Lange SS, Tomida J, Boulware KS, Bhetawal S, Wood RD. The Polymerase Activity of Mammalian DNA Pol ζ Is Specifically Required for Cell and Embryonic Viability. PLoS Genet. 2016 Jan 4;12(1):e1005759.
Section of testis from POLN deficient mouse
We have discovered POLN as a homologue of POLQ and are working to identify its biochemical activities. The POLN gene is predominantly expressed in
testis and encodes a protein able to displace DNA strands and bypass DNA damage.
The image shows gamma-H2AX foci (green dots), which represent sites of DNA damage, in spermatid cells lacking POLN. In this example, DNA damage was induced by x-ray irradiation
Takata KI, Reh S, Yousefzadeh MJ, Zelazowski MJ, Bhetawal S, Trono D, Lowery MG, Sandoval M, Takata Y, Lu Y, Lin K, Shen J, Kusewitt DF, McBride KM, Cole F Wood RD. (2017) Analysis of DNA polymerase ν function in meiotic recombination, immunoglobulin class-switching, and DNA damage tolerance. PLoS Genet. 2017 Jun 1;13(6):e1006818.
DNA Helicase HELQ
Agents that cause interstrand DNA crosslinks (ICL) are widely used to treat cancer. Our work found that the DNA helicase HELQ helps defend human cells against ICL- inducing agents. HELQ associates with the ATR-ATRIP complex that is important for cellular response to replication stress and DNA damage. HELQ also associates with signaling and homologous recombination components such as BCDX2, a RAD51 paralog, which is sometimes altered in ovarian cancer.
In the image, the red, jagged line outlined in gray represents an interstrand crosslink. HELQ, along with ATR and RAD51 paralogs, helps cells tolerate these crosslinks.
Takata K, Reh S, Tomida J, Person MD, Wood RD. . Human DNA helicase HELQ participates in DNA interstrand crosslink tolerance with ATR and RAD51 paralogs. Nat Commun. 2013;4:2338.